The chlamydiae are among the most common animal pathogens in the world. These Gram-negative cells are unusual among bacteria in being obligate intracellular organisms. They replicate within the infected host cell and, lacking enzymes able to produce their own energy from metabolic reactions, rob their hosts of energy by using ATP produced by the host for their own requirements. Chlamydia trachomatis is one of the three species classifications of the genus Chlamydia, and is a human pathogen. See American Society for Microbiology, Manual of Clinical Microbiology (5th ed. 1991). Chlamydia trachomatis strains include the causal agents of trachoma, inclusion conjunctivitis, and genital tract diseases. In the latter context, C. trachomatis is the leading cause of sexually transmitted disease in the world, causing urethritis in men and cervicitis in women. An infected woman may transmit the infection to her child during birth, resulting in pneumonia or eye disease among other conditions. Early detection of C. trachomatis infection in affected individuals can accelerate necessary treatment and prevent continued transmission of the agent.
It is therefore an object of the present invention to provide nucleic acid hybridization probes for the rapid and specific detection of C. trachomatis in test samples and particularly in human clinical specimens.
As used herein, the term "test sample" is intended to mean any sample suspected of containing the intended target nucleic acid, and includes but is not limited to: biological samples, body fluids or exudate such as urine, blood, milk, cerebrospinal fluid, sputum, saliva, stool, lung aspirates, throat or genital swabs, clinical specimens containing one or more of the foregoing, environmental samples, food samples and laboratory samples.
Nucleic acid hybridization is the process by which two nucleic acid strands having completely or partially complementary nucleotide sequences come together under predetermined reaction conditions to form a stable, double-stranded hybrid with specific hydrogen bonds. Either nucleic acid strand may be a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA); thus hybridization can involve RNA:RNA hybrids, DNA:DNA hybrids, or RNA:DNA hybrids.
Thus, as used in this application, the term "hybridization" refers to the ability of two completely or partly complementary single nucleic acid strands to come together in an antiparallel orientation to form a stable structure having a double-stranded region. The two constituent strands of this double-stranded structure, sometimes called a hybrid, are held together with hydrogen bonds. Although these hydrogen bonds most commonly form between nucleotides containing the bases adenine and thymine or uracil (A and T or U) or cytosine and guanine (C and G), base pairing can form between bases who are not members of these "canonical" pairs. Non-canonical base pairing is well-known in the art. See e.g., The Biochemistry of the Nucleic Acids (Adams et al., eds., 1992).
Nucleic acid hybridization is a common method for detecting and quantitating target nucleic acids having specific nucleotide sequences. Such methods are useful for identifying and classifying organisms, diagnosing infectious diseases and genetic abnormalities, testing food and drugs, and identifying criminal suspects, among numerous other things. Typically, nucleic acid hybridization assays use a labeled oligonucleotide hybridization assay probe having a nucleic acid sequence complementary to the target sequence. Such labels are well known in the art, and may include radioactive isotopes, enzymes, or fluorescent, luminescent, or chemiluminescent groups; the Applicants prefer the use of chemiluminescent acridinium esters as labels. See Arnold et al., U.S. Pat. No. 5,185,439, which enjoys common ownership with the present application and is incorporated by reference herein. The probe is mixed with a sample suspected of containing a nucleic acid having the target sequence under hybridization conditions suitable for allowing annealing of the two strands by hydrogen bonding in the region of complementarity. The probe then hybridizes to the target nucleic acid present in the sample. The resulting hybrid duplex may be detected by various techniques well known in the art, such as hydroxyapatite adsorption. Also included among these techniques are those that involve selectively degrading the label present on unhybridized probe and then measuring the amount of label associated with the remaining hybridized probe, as disclosed in Arnold et al., U.S. Pat. No. 5,283,174, which enjoys common ownership with the present application and is incorporated by reference herein. This latter technique, called the hybridization protection assay (HPA), is presently preferred by the Applicant.
Often a test sample will not contain a great enough number of nucleic acid molecules to permit direct detection or quantification by nucleic acid hybridization due to the sensitivity limits of the particular label used. In such a case, the amount of detectable target nucleotide sequence is increased before nucleic acid hybridization is used to identify its presence or amount in the test sample. This procedure is termed nucleic acid amplification, and the method of increasing the amount of the target nucleic acid is referred to as amplifying the target nucleic acid or target nucleotide sequence.
Amplification methods involve the use of at least one nucleic acid strand containing a target nucleotide sequence as a template in a nucleic acid polymerizing reaction to produce a complementary second strand containing the target nucleotide sequence. By repeating this process, using the product nucleic acids as templates in subsequent cycles, the number of nucleic acid molecules having the target nucleotide sequence increases rapidly.
A number of amplification methods have been described; among these are various embodiments of the polymerase chain reaction (PCR), (see e.g., Mullis et al., U.S. Pat. No. 4,683,195), and methods which utilize in vitro transcription (RNA synthesis) in one or more step of the procedure, (see e.g., Murakawa et al., DNA 7:287-295, Burg et al., PCT Application No. WO89/1050, Gingeras et al., PCT Application No. WO88/10315, Kacian & Fultz, European Application No. 89313154, McDonough, et al., PCT Publication No. WO 94/03472, Kacian, et al., PCT Publication No. WO 93/22461, and Dattagupta, et al. (filed in the United States Mar. 16, 1994, U.S. application Ser. No. 08/215,081). The disclosure of these references are incorporated by reference herein; the last two of these references enjoy common ownership with the present application.
Most nucleic acid amplification methods employ oligonucleotide primers and/or promoter-primers. These primers or promoter-primers are relatively short,(preferably between 10 and 100 nucleotides; most preferably between about 12 and 50 nucleotides in length) single-stranded nucleic acid molecules which are chemically, biologically or enzymatically synthesized, designed, and/or selected through human intervention to have a nucleotide sequence complementary to at least a portion of a nucleotide sequence region of the intended target nucleic acid. When the primer or promoter-primer is brought together with the target nucleic acid under conditions which allow the two nucleic acid strands to hybridize, at least part of the primer or promoter-primer forms a double-stranded, hydrogen-bonded hybrid with the target nucleic acid. Often, but not invariably, a distinctive feature of such a hybrid is that a primer or promoter-primer has a free 3' hydroxyl group able to react with a nucleotide in a nucleic acid polymerase-mediated primer extension reaction while hybridized. However, a free 3' hydroxyl group may not be necessary for a promoter-primer to function as a promoter.
A primer extension reaction occurs when the double-stranded primer:target nucleic acid hybrid is contacted with a nucleic acid polymerase, and the necessary nucleotide triphosphates. The primer's available 3' hydroxyl group enables the nucleic acid polymerase to specifically begin adding nucleotide residues to the 3' end of the primer; hence, the nascent nucleic acid strand grows in the 5' to 3' direction relative to the primer's polarity. The sequence of the growing primer extension product is dictated by the nucleotide sequence of the target nucleic acid template. Thus, the primer initiates the synthesis of a complementary nucleic acid strand in the region of initial annealing or hybridization.
A "promoter-primer" can function as a primer, in that it has a 3' region of complementarity to its intended nucleic acid target, when it has a free 3' hydroxyl group. Additionally, a promoter-primer has a nucleotide sequence region at its 5' end which is not complementary to the target nucleic acid. When this region is made double-stranded through the action of a nucleic acid polymerase (this time extending the 3' end of the template nucleic acid), the double-stranded non-complementary region can function as an initiation site for RNA synthesis using an enzyme having RNA polymerase activity.
Depending on the uniqueness of the target nucleotide sequence and the degree of selectivity desired in a hybridization assay, a primer or promoter-primer may also or alternatively function as a hybridization assay probe. Alternatively, a hybridization assay probe or amplification oligonucleotide may be designed and used solely for its primary function.
A hybridization assay probe is used to detect and/or quantify the presence of the intended target nucleic acid; such a probe is usually labeled with a radioactive or luminescent atom or a detectable chemical group, such as a chemiluminescent moiety. The Applicant prefers using acridinium ester derivatives as a labeling reagent. Sometimes the intended target nucleic acid will include any of a population of different nucleic acid molecules with nucleotide sequences usually derived from a biological source. By way of example only, and not of limitation, the target nucleotide sequence may be shared by the nucleic acids of a genus of organisms (but not by organisms outside the genus) the detection of any of which is desired. Alternatively, the target nucleotide sequence may be unique to a species of organism or to a strain of that species.
Not all probes are necessarily labeled. Some hybridization probes, termed "helper oligonucleotides" or "helper probes", are designed to facilitate the ability of a separate labeled probe to bind to its target nucleotide sequence. Although not wishing to be bound by theory, helper probes are thought to facilitate binding of the labeled probe by locally decreasing the amount of intramolecular hydrogen-bonding in the target nucleic acid, thus making the target nucleotide sequence more available for specific hybridization with the labeled probe. Depending on the location of the labeled probe's binding site and the secondary structure of the target nucleic acid, helper probes may be directed to nucleotide sequence regions proximal to the labeled probe's binding site, or directed to regions distal from the binding site which nevertheless affect probe binding. Helper probes are described in Hogan et al., U.S. Pat. No. 5,030,557 which enjoys common ownership with the current application, and which is incorporated by reference herein.
Descriptions of the use of nucleic acid hybridization to detect the presence of particular nucleic acid sequences are given in Kohne, U.S. Pat. No. 4,851,330 and in Hogan et al., International Patent Application No. PCT/U.S. Ser. No. 87/03009; both of these references enjoy common ownership with the present application, and are incorporated by reference herein. Hogan describes methods for determining the presence of a non-viral organism or a group of non-viral organisms in a sample (e.g., sputum, urine, blood and tissue sections, food, soil and water) using nucleic acid hybridization techniques.
Hogan, supra, also describes a number of hybridization probes which specifically detect only targeted ribosomal RNA (rRNA) nucleotide sequences belonging to a specific organism or group of organisms.
DNA hybridization assay probes for detection of C. trachomatis have been described. Hyppia et al., J. Gen. Microbiol.130:3159-64 (1984), describe the isolation of a 6.7 kb plasmid from C. trachomatis and its use as a hybridization probe. Griffais et al., Res. Microbiol. 140:139-141 (1989), Ostergaard, et al., J. Clin. Microbiol. 28:1254-1260 (1990), McGarity et al., Gut 32:1011-1015 (1991), Claas et al., J. Clin. Microbiol. 29:42-45 (1991), Longiaru, EPO 420 260, Application No. 90118620.5, and Longiaru et al., U.S. Pat. No. 5,232,829 amplified C. trachomatis plasmid nucleotide sequences using the polymerase chain reaction (PCR) in conjunction with amplification oligonucleotides. Amplification of sequences encoding the major outer membrane protein (MOMP) of C. trachomatis was described by Dutilh et al., Res. Microbiol. 140:7-16 (1989), Holland et al., J. Infec. Dis. 62:984-987 (1990), Bobo et al., J. Clin. Microbiol. 28:1968-197 (1990), Holland et al., Infec. Immun. 60:2040-2047 (1992), and Palmer et al., J. Clin. Pathol. 44:321-325 (1991). Ossewaarde et al., J. Clin. Microbiol. 30:2122-2128 (1992) and Roosendaal et al., J. Med. Microbiol. 38:426-433 (1993) describe amplification of plasmid and MOMP sequences.
Hogan et al., PCT Application Number PCT/U.S. Ser. No.87/03009, which enjoys common ownership with the present application, and Shah et al., PCT Publication Number WO90/15159, describe probes for the detection of C. trachomatis 16S and 23S rRNA sequences. Naher et al., Genitourin. Med. 65:319-322 (1989), Kluytmans et al., J. Clin. Microbiol. 29:2685-2689 (1991), Scieux et al., Res. Microbiol. 143:755-765 (1992), and Holland et al., Infect. Immun., supra, describe the use of probes directed to C. trachomatis rRNA. Cheema et al., Amer. J. Med. Sci. 302:261-268 (1991) describe probes directed to Chlamydia ribosomal DNA. Pollard et al., Mol. Cell. Probes 3:383-389 (1989), Roosendaal, supra, and Claas et al., Eur. J. Clin. Microbiol Infec. Dis. 9:864-868 (1990) describe the amplification of nucleotide sequences derived from the 16S ribosomal subunit of Chlamydia species.